Microdialysis Sampling Extraction Efficiency of 2-Deoxyglucose: Role

Microdialysis Sampling Extraction Efficiency of. 2-Deoxyglucose: Role of Macrophages in Vitro and in Vivo. Xiaodun Mou and Julie A. Stenken*. Departme...
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Anal. Chem. 2006, 78, 7778-7784

Microdialysis Sampling Extraction Efficiency of 2-Deoxyglucose: Role of Macrophages in Vitro and in Vivo Xiaodun Mou and Julie A. Stenken*

Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180-3590

Macrophages are a class of inflammatory cells believed to direct the outcome of device biocompatibility. Despite their relevance to implanted in vivo devices, particularly implanted glucose sensors, few studies have attempted to elucidate how these cells affect device performance. Microdialysis sampling probes were used to determine glucose uptake alterations in the presence of resting and activated macrophages in vitro. Significant differences for 2-deoxyglucose (2-DG) relative recovery at 1.0 µL/min were observed between resting (74 ( 7%, n ) 18) and lipopolysaccharide (LPS) (1 µg/mL)-activated (56 ( 6%, n ) 18) macrophages in culture that had 2-DG spiked into the media (p < 0.005). To establish if in vitro characterization could be correlated to in vivo studies, microdialysis probes were implanted into the dorsal subcutis of male Sprague-Dawley rats for 0, 3, 5, and 7 days. An internal standard, 2-DG, was passed through the microdialysis probe during in vivo studies. No significant differences in 2-DG extraction efficiency from the probe into the tissue site were observed in vivo among microdialysis probes implanted into the subcutaneous space of Sprague-Dawley rats for either 3, 5, or 7 days vs probes implanted the day of sample collection. These results suggest that macrophage activation in vivo at implant sites is much lower than highly activated macrophages in vitro. It is important to note that these results do not rule out the potential for increased glucose metabolism at sensor implant sites. The creation of reliable implantable glucose sensors for continuous glucose measurements in diabetic humans has been an active research topic for more than 3 decades.1 Extensive research efforts have focused on solving many of the challenging problems associated with in vivo glucose measurements.2,3 Some problems, such as removal of electrochemical interferences (e.g., acetaminophen, ascorbate), have been overcome with appropriate electrode design.4,5 Other concerns, such as direct long-term integration with the host have not been completely solved.6-9 * To whom correspondence should be addressed. Phone: 518-276-2045. Fax: 518-276-4887. Email: [email protected]. (1) Clark, L. C.; Sachs, G. Ann. N.Y. Acad. Sci. 1968, 148, 133-153. (2) Wilson, G. S.; Hu, Y. Chem. Rev. 2000, 100, 2693-2704. (3) Heller, A. Annu. Rev. Biomed. Eng. 1999, 1, 153-175.

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The primary challenge affecting subcutaneous implanted glucose sensors as well as any implanted sensing device has been overcoming the host foreign body response while retaining proper function, for example, resistance to signal drift and calibration maintenance.10,11 The immense tissue-integration challenges associated with in vivo glucose measurements have precluded the long-term implantation (1-4 weeks) of glucose sensors for continuous glucose monitoring.12-14 Device implantation creates a wound, resulting in an activation of complex biochemical and cellular mechanisms to heal the injury, which is referred to as the foreign body response (Figure 1).15,16 Research aimed to improve the integration of sensors with their host and thus control the foreign body response has focused on chemical release strategies for nitric oxide and vascular endothelial growth factor (VEGF).17,18 Among the different foreign body response mechanisms, fibrotic capsule formation and macrophage recruitment are among the most likely to cause variations in glucose sensor performance (e.g., result in lag times, differences in day-to-day sensitivity, and sensor drift), as denoted in Figure 1.19-21 The fibrotic encapsulation (4) Zhang, Y.; Hu, Y.; Wilson, G. S.; Moatti-Sirat, D.; Poitout, V.; Reach, G. Anal. Chem. 1994, 66, 1183-1188. (5) Chen, T.; Friedman, K. A.; Lei, L.; Heller, A. Anal. Chem. 2000, 72, 37573763. (6) Gifford, R.; Kehoe, J. J.; Barnes, S. L.; Kornilayev, B. A.; Alterman, M. A.; Wilson, G. S. Biomaterials 2006, 27, 2587-2598. (7) Wilson, G. S.; Gifford, R. Biosens. Bioelectron. 2005, 20, 2388-2403. (8) Long, N.; Yu, B.; Moussy, Y.; Moussy, F. Diabetes Technol. Ther. 2005, 7, 927-936. (9) Ahmed, S.; Dack, C.; Farace, G.; Rigby, G.; Vadgama, P. Anal. Chim. Acta 2005, 537, 153-161. (10) Wisniewski, N.; Moussy, F.; Reichert, W. M. Fresenius’ J. Anal. Chem. 2000, 366, 611-621. (11) Ratner, B. D. J. Controlled Release 2002, 78, 211-218. (12) Gilligan, B. J.; Shults, M. C.; Rhodes, R. K.; Jacobs, P. G.; Brauker, J. H.; Pintar, T. J.; Updike, S. J. Diabetes Technol. Ther. 2004, 6, 378-386. (13) Pickup, J. C.; Hussain, F.; Evans, N. D.; Sachedina, N. Biosens. Bioelectron. 2005, 20, 1897-1902. (14) Choleau, C.; Klein, J. C.; Reach, G.; Aussedat, B.; Demaria-Pesce, V.; Wilson, G. S.; Gifford, R.; Ward, W. K. Biosens. Bioelectron. 2002, 17, 641-646. (15) Anderson, J. M. Annu. Rev. Mater. Res. 2001, 31, 81-110. (16) Koenig A. L; Grainger D. W. In Biomimetic Materials and Design; Dillow, A.K., Lowman, A.M., Eds.; New York: Marcel Dekker: Inc., 2002; p 187250. (17) Gifford, R.; Batchelor, M. M.; Lee, Y.; Gokulrangan, G.; Meyerhoff, M. E.; Wilson, G. S. J. Biomed. Mater. Res. 2005, 75A, 755-766. (18) Ward, W. K.; Wood, M. D.; Casey, H. M.; Quinn, M. J.; Federiuk, I. F. Diabetes Technol. Ther. 2004, 6, 137-145. (19) Woodward, S. C. Diabetes Care 1982, 5, 278-281. (20) Clark, L. C.; Spokane, R. B.; Homan, M. M.; Sudan, R.; Miller, M. ASAIO Transactions 1988, 34, 259-265. 10.1021/ac061124i CCC: $33.50

© 2006 American Chemical Society Published on Web 10/20/2006

Figure 1. Foreign body response progression with the role of the microdialysis probe in this study.

around the sensor causes response delays and decreased signal due to analyte diffusional lag through the capsule to the sensor. Macrophage activation at the site of implanted glucose sensors is poorly understood due to the lack of analytical methods to locally interrogate these cells. Yet, the importance of macrophages toward orchestrating the overall foreign body response has been well-documented in the biomaterials literature for 20 years.22-24 Macrophages help to remodel the wound site via release of different chemical signaling agents (cytokines) and enzymes (e.g., various matrix metalloproteinases) and can remain at the implant site throughout its lifetime. Microdialysis sampling is a diffusion-based separation process that has been widely used for sampling from complex matrixes in vitro and in vivo.25-27 The calibration of a microdialysis probe is obtained via its extraction efficiency (EE). The steady-state equation for extraction efficiency

EE )

Cinlet - Coutlet ) Cinlet - Csample 1 - exp

(

)

-1 (1) Qd(Rd + Rm + RECF + Rtr)

has been derived by Bungay et al. and is shown in eq 1.28,29 In this equation, Coutlet is the analyte outlet concentration, Cinlet is the analyte inlet concentration, and Csample is the analyte concentration far from the probe. When Cinlet equals 0, EE becomes the (21) Rebrin, K.; Steil, G. M. Diabetes Technol. Ther. 2000, 2, 461-472. (22) Anderson, J. M.; Miller, K. M. Biomaterials 1984, 5, 5-10. (23) Rhodes, N. P.; Hunt, J. A.; Williams, D. F. J. Biomed Mater. Res. 1997, 37, 481-488. (24) Labow, R. S.; Meek, E.; Santerre, J. P. Biomaterials 2001, 22, 3025-3033. (25) Weintjes, K. J.; Vonk, P.; Vonk-Van Klein, Y.; Schoonen, A. J. M.; Kossen, N. W. Diabetes Care 1998, 21, 1481-1488. (26) Hillered, L.; Persson, L. Scand. Cardiovasc. J. 2003, 37, 13-17. (27) de la Pena, A.; Liu, P.; Derendorf, H. Adv. Drug Delivery Rev. 2000, 45, 189-216. (28) Bungay, P. M.; Morrison, P. F.; Dedrick, R. L. Life Sci. 1990, 46, 105-19. (29) Bungay, P. M.; Newton-Vinson, P.; Isele, W.; Garris, P. A.; Justice, J. B. J. Neurochem. 2003, 86, 932-946.

ratio of Coutlet to Csample, which has been termed relative recovery (RR). EE is independent of analyte directional movement to or from the dialysis probe and can be used when analytes are either recovered or delivered through the probe. However, for clarity in this work, all experiments performed in the “recovery” mode, during which analyte is moving from the sample into the dialysis probe, will be ascribed RR values. Experiments performed in the “delivery” mode, during which analyte is infused through the probe and extracted into the sampling media, will be denoted as “EE.” Microdialysis sampling EE is dependent on flow rate (Q) and diffusional mass transport resistances through the dialysate (Rd), membrane (Rm), tissue space (RECF), and any trauma layer near the probe (Rtr). For any analyte, its EE is highly sensitive to the kinetic processes occurring within the sample media external to the microdialysis probe.30,31 The mass transport resistance of the tissue is greatly affected by the kinetics of analyte uptake including metabolism (km) and capillary permeability, that is, efflux from ECF to plasma (kep) and uptake from plasma to ECF (kpe) as denoted in eqs 2 and 3.28,32 In these equations K0 and K1 are Bessel functions that typically arise in steady-state solutions to diffusion and kinetic mass transfer problems in cylindrical coordinates (Bessel functions can be evaluated using standard software programs, such as Microsoft Excel, Matlab, etc).

RECF )

Γ[K0(ro/Γ)/K1(ro/Γ)] 2πroLDeφe

Γ ) xDe/(kxep + kre + krc)

(2) (3)

The description for Γ is defined as a “combined loss rate constant”, which is a composite function of the analyte diffusion coefficient (30) Robinson, T. E., Justice, J. B., Eds. Microdialysis in the Neurosciences; Elsevier: Amsterdam, 1991. (31) Stenken, J. A.; Lunte, C. E.; Southard, M. Z.; Ståhle, L. J. Pharm. Sci. 1997, 86, 958-966. (32) Stenken, J. A. Anal. Chim. Acta 1999, 379, 337-357.

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within the media external to the probe (De) and rate constants of the capillary exchange (kxep), metabolism of analyte in the ECF (kre), and intra- to extracellular exchange (krc). In addition to all the rate constants, the outer membrane radius of the microdialysis probe (ro), the length of the probe (L), and the volume fraction in the ECF (φe) also attribute to the mass transport resistance through the tissue space. Knowing that microdialysis sampling extraction efficiency depends on the analyte removal kinetics external to the microdialysis probe as denoted in eq 3 allows the possibility to test whether significant alterations in glucose uptake affect microdialysis EE either in vitro or in vivo. In this work, the bidirectional diffusive nature of microdialysis sampling was used to determine the effect of glucose uptake kinetic alterations between active and resting macrophages in cell culture as well as in vivo on the dialysis probe calibration (EE or RR) for both glucose and the internal standard 2-deoxyglucose (2-DG), as outlined in Figure 1. 2-DG is routinely used as a tracer of glucose uptake, since it is not metabolized by phosphoglucoisomerase during glycolysis.33 Lipopolysaccharide (LPS) was used to stimulate macrophages into an inflammatory state. It has been reported that activated macrophages have greater glucose uptake kinetics when compared to normal tissue cells.34-37 If glucose uptake kinetic rates were increased during macrophage activation, this would be reflected as an increase in 2-DG EE% (loss from the perfusion fluid) during the microdialysis sampling process. Since activated inflammatory cells are known to be at the site of implants, including sensor implants, changes in glucose uptake kinetics may affect localized glucose sensing. EXPERIMENTAL SECTION Chemicals. Water (HPLC grade, Fisher Scientific, Fair Lawn, NJ) was used to prepare all the solutions. Glucose (Sigma, St. Louis, MO) and 2-deoxyglucose (99%, Acros Organics, NJ) were used as standards for calibration of the microdialysis probes and detection system. All the samples were prepared or diluted in a phosphate-buffered saline solution (PBS, pH 7.2 consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4). Potassium chloride, potassium phosphate monobasic, sodium chloride, and sodium phosphate dibasic were purchased from Fisher Scientific, Fair Lawn, NJ. For macrophage cell culture, the media was a mixture of Dulbecco’s Modification of Eagle’s Medium (DMEM) (Fisher Scientific, Pittsburgh, PA), fetal bovine serum (FBS, Biowhittaker, Walkersvile, MD), and penicillin and streptomycin (Fisher Scientific, Pittsburgh, PA). The DMEM used in the general cell culture has a reported glucose concentration of 4.5 g/L (∼25 mM). During the microdialysis experiment, DMEM with a glucose concentration of 1.0 g/L was used (∼5 mM). Trypan blue solution (0.4%) purchased from Sigma (St. Louis, MO) was used as a dye to count the number of viable cells before and after each cell culture experiment. Bacterial lipopolysaccharide (Escherichia coli, K-23; Sigma) was employed to induce the macrophages into an inflammatory state. The mobile phase of the detection system contained barium acetate (Ba(OAc)2, 99%, Fisher Scientific, Pittsburgh, PA) and sodium (33) Wick, A. N.; Drury, D. R.; Nakada, H. I.; Wolfe, J. B. J. Biol. Chem. 1957, 224, 963-969. (34) Hamilton, J. A.; Vairo, G.; Lingelbach, S. R. J. Cell. Physiol. 1988, 134, 405412. (35) Meszaros, K.; Lang, C. H.; Bagby, G. J.; Spitzer, J. J. FASEB J. 1988, 2, 3083-3086. (36) Lang, C. H.; Dobrescu, C. Metab., Clin. Exp. 1991, 40, 585-593. (37) Fernandes, L. C.; Mattozo, C. A.; Machado, U. F.; costa Rosa, L. F. B. P.; Curi, R. Cell Biochem. Funct. 1996, 14, 187-192.

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hydroxide (NaOH) (Mallinckrodt Baker, Inc. Paris, KY). Sterile water (MP Biomedicals, Inc. Solon, OH) was used to perfuse microdialysis probes before implantation in vivo. Ion-Exchange Chromatography with Pulsed Amperometric Detection (IC-PAD). Glucose and 2-DG in microdialysates and the cell culture media were directly separated by anionexchange chromatography, followed by quantification with pulsed amperometric detection.38-40 The IC-PAD system consisted of an LC-10AD pump (Shimadzu) and an amperometric detector from Antec Leyden (DECADE, Antec Leyden, The Netherlands) that contains an internal column heater. An anion-exchange column, Dionex CarboPac PA1, 250 × 2 mm, with guard column (50 × 2 mm) was used for carbohydrate separation. The flow-through detection cell (Antec Leyden VT-03) contained a gold working electrode and Ag/AgCl reference electrode. Pulsed amperometric detection was carried out using the following pulse potentials and time durations: EDET ) +0.10 V (tDET ) 600 ms), EOX ) 0.80 V (tOX ) 200 ms), ERED ) -0.60 V (tRED ) 200 ms). The PAD detector and columns were maintained at 30 °C in an internal column oven. Each day, the ion-exchange columns were regenerated for 30 min with a 200 mM sodium hydroxide solution. A solution with 10 mM sodium hydroxide and 2 mM barium acetate was used as the mobile phase. The barium acetate was added to the alkaline solution the day before the experiment to remove excess carbonate via the precipitation of the barium carbonate salt.41 The mixture was filtered through a 0.2-µm filter (Whatman International Ltd., Maidstone, England) before use. This approach removes unwanted carbonate, which improves run-to-run retention time reproducibility. The mobile phase was sparged with argon at low flow rates during analyses. All separations were carried out using an isocratic elution with a 0.25 mL/min flow rate. Concentrations in the dialysates were predicted by comparing peak area to standards of 2-DG and glucose in the range of 0-100 µM in PBS. A calibration curve was performed on each working day. The microdialysates were diluted 100 times with PBS solution, then separated and analyzed using the IC-PAD system with the same parameters as those for the standards. Cell Culture. RAW 264.7 murine macrophage cells were obtained as a gift from Professor Michelle Lennartz of Albany Medical College. The culture media (90 v/v % DMEM and 10 v/v % FBS) was supplemented with penicillin (1000 U/mL) and streptomycin (100 µg/mL). The macrophage cells were incubated at 37 °C with 5% CO2 using standard cell culture methods. Macrophages (1 × 106/mL) placed in 2.8 mL of cell culture media in the well of a 24-well plate (Tissue culture treated, Corning, NY) were stabilized overnight in DMEM (25 mM glucose, 10% FBS) to allow for cell attachment to the culture plate. On the day of sample collection, fresh media was added to used to replace the overnight media in each well. Microdialysis System. A BAS Bee microdialysis pump with a three-syringe bracket and 1-mL syringes (Bioanalytical System Inc., West Lafayette, IN) was used for all microdialysis experiments. Commercially available CMA/20 microdialysis probes consisting of a polycarbonate/polyether (PC) membrane, 10-mm length and 20-kDa molecular weight cutoff (MWCO) (CMA Microdialysis, Inc., North Chelmsford, MA), were immersed into (38) Rakotomanga, S.; Baillet, A.; Pellerin, F.; Baylocq-Ferrier, D. J. Pharm. Biomed. Anal. 1992, 10, 587-591. (39) Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990, 62, 589A-597A. (40) Zook, C. M.; LaCourse, W. R. Anal. Chem. 1998, 70, 801-806. (41) Cataldi, T.; Campa, C.; Angelotti, M.; Bufo, S. A. J. Chromatogr., B 1999, 855, 539-550.

the culture wells containing previously described designed tops.42 Unless otherwise stated, a PBS solution was used as the perfusion fluid and was infused with a 1.0 µL/min flow rate. All the experiments with microdialysis probes in cell culture media were performed in a cell culture incubator at 37 °C with 5% CO2. The dialysates were collected into 200-µL microcentrifuge tubes sealed with Parafilm to prevent solvent evaporation. The solvent loss during the sample collection period was determined to be 0.3 ( 0.1% (n ) 10) when comparing the weight of the dialysates with the sample media of the same volume. Lipopolysaccharide Affect on Glucose Concentrations in Macrophage Culture. The macrophages (1 × 106/mL) in DMEM (10% FBS) with a reported glucose concentration of 25 mM were placed in 6 wells of a 24-well cell culture plate. Serial doses of LPS (1 ng/mL, 10 ng/mL, 100 ng/mL, 1 µg/mL, and 10 µg/mL) were added into wells B, C, D, E, and F, separately. The well denoted “A” was the control and contained no LPS. Aliquots (50 µL) were taken from each well immediately after LPS addition and at 4, 8, 20, and 24 h. Each aliquot was mixed with an equal volume of 10% (v/v) perchloric acid and placed in the refrigerator at 4 °C for 10 min. The mixture was centrifuged and diluted with PBS by 250-fold. The diluted samples were analyzed using the IC-PAD system. Alteration of 2-Deoxyglucose Microdialysis EE by Macrophages. During each microdialysis sampling experiment, one culture well contained macrophages that served as a control (no LPS), and the other well contained macrophages to which LPS was added. To each well, 2-DG (5 mM) was added to the media containing 5 mM glucose. Microdialysis sampling procedures commenced 1 h after the LPS addition, and samples were collected every 30 min for 3 h. During the microdialysis sampling process, the concentrations of 2-DG and glucose in the sample media were obtained by directly analyzing an aliquot (50 µL) from the media. After the experiments, the average density of the viable macrophages in the LPS-activated wells was 0.95 ( 0.16 × 106 cells/ mL (n ) 3), as determined by Trypan Blue staining. In Vivo Experiments. Male Sprague-Dawley rats (250-300 g, Taconic, Taconic, NY) were used for all in vivo experiments. The rats had free access to food and water with a 12-h on/off light cycle prior to the experiments. The rats were anesthetized with isofluorane and kept warm using a temperature-controlled heating pad (CMA Microdialysis, North Chelmsford, MA) during surgery and sample collection. All surgical procedures were performed using aseptic technique. The Albany Medical College IACUC committee has approved these procedures. Prior to implantation, the probes were removed from their sealed packages and perfused with sterile water in 70% ethanol in a biosafety cabinet to remove the glycerol that impregnates the membrane to keep it viable during shipping. For acute implantations, two 10-mm CMA/20 microdialysis probes with a polycarbonate/polyether membrane (20 kDa MWCO) were implanted into each side of the dorsal subcutis of the rats. A perfusion fluid containing 2-DG (5 mM in PBS) was infused through both microdialysis probes with a flow rate of 5 µL/min for 5 min following probe implantation. After this 5-min flushing period, the flow rate was reduced to 1 µL/min. Following a 15-minute equilibration time at the 1 µL/min flow rate, the dialysates were then collected every 30 min for 3 h while the animal was maintained under anesthesia. The dialysates were stored at 4 °C in the refrigerator before dilution and analysis. All (42) Sun, L.; Stenken, J. A. J. Chromatogr., B 2003, 796, 327-338.

Table 1. 2-Deoxyglucose and Glucose Microdialysis Sampling RR%a n sample media

2-DG

glucose

probe

sample

PBS culture media without FBS culture media (1% FBS)

70 ( 5 76 ( 6 78 ( 9

71 ( 5 75 ( 4 74 ( 5

3 3 3

4 4 4

a Microdialysis sampling was performed at 1.0 µL/min with PBS buffer at 37 °C. All media contained 5 mM 2-DG and glucose. Samples were collected for 30 min over a 2-h period. Data represent mean ( SD averaged among all samples collected in each type of media (n ) 12).

samples were analyzed no later than 48 h after sample collection. For chronic implantations, one PC probe was implanted on the right side of the dorsal subcutaneous space of rats for up to 3, 5, or 7 days. On the day of dialysate collection, a second PC probe was implanted into the left side of the dorsal subcutaneous space. The remainder of the experimental protocol for sample collection and analysis followed that for the acute implantation procedure described above. RESULTS AND DISCUSSION Separation of Glucose and 2-DG. To determine retention time reproducibility, glucose and 2-deoxyglucose solutions (25 µM) were prepared with PBS, then separated and analyzed by the ion chromatography-pulsed amperometric system. Sample injections (10 µL) were performed every 15 min over a 6-h period. The retention times were 5.27 ( 0.10 and 3.76 ( 0.05 min (n ) 24) for glucose and 2-DG, respectively. The k′ of glucose was 2.0 ( 0.05, and that of 2-DG was 1.2 ( 0.02 (n ) 24), which illustrates the retention reproducibility for these IC-PAD conditions.43 Microdialysis Sampling of Glucose and 2-Deoxyglucose in Different Sample Media. Microdialysis probes were tested in different media to determine if protein from the culture media would affect extraction efficiency for either 2-DG or glucose. Table 1 shows the resulting RR% values obtained among three separate media (PBS, culture media without fetal bovine serum (FBS), and culture media with 1% FBS). Among these three different media, RR% values were roughly 70% and were not different. Macrophage Activation. A range of LPS concentrations between a blank (no LPS) and 10 µg/mL were applied to the macrophage-containing wells to determine the level at which glucose consumption was significantly increased. Table 2 shows the resulting glucose concentrations obtained from the individual wells and measured over a 24-h period. After 4 h, there were significant differences between the glucose concentrations in wells with LPS and those of the blank. Macrophage cultures with either 1 or 10 ng/mL LPS exhibited similar glucose concentrations (∼18 mM) at 4 h. Glucose concentrations obtained from macrophages given the doses ranging from 100 ng/mL to 10 µg/mL were less than those obtained for the 1 and 10 ng/mL dose. On the basis of these experiments, a concentration of 1 µg/mL of LPS was chosen for the remaining cell culture experiments. 2-Deoxyglucose and Glucose RR in Macrophage Cell Cutlure. Microdialysis sampling recovery (RR%) values for 2-DG and glucose were significantly different between resting and LPSactivated macrophages (Table 3). Table 3 shows the individual (43) Andreas, G.; Andreas, S. Microchim. Acta 2004, 146, 97-102.

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Table 2. Glucose Concentration (mM) after Lipopolysaccharide Addition to Cultured Macrophagesa LPS dose

0

4h

8h

20 h

24 h

blank 1 ng/mL 10 ng/mL 100 ng/mL 1 µg/mL 10 µg/mL

23.1 23.0 22.8 22.5 22.8 22.6

21.5 17.5 18.0 15.3 13.9 14.3

15.8 17.4 16.4 13.9 14.9 13.1

15.3 15.4 14.0 10.4 14.2 13.1

14.6 14.0 9.9 8.6 9.4 8.6

a Macrophages (1 × 106 /mL) in DMEM culture media with a reported glucose concentration of 4.5 g/L (25 mM) were placed into individual wells of a 24-well cell culture plate. Individual aliquots (50 µL) were removed and analyzed at the specified time periods.

statistics for glucose and 2-DG microdialysis sampling RR% and concentration values obtained from aliquots of the culture media at 30-min increments within the macrophage culture wells. The overall average among all the data points showed significant decreases in 2-DG (56 ( 6%) and glucose (61 ( 9%) RR% for the active macrophages as compared with those (2-DG, 74 ( 7%; glucose, 74 ( 7%) for resting macrophages. For 2-DG, a clear reduction in RR% in the activated vs resting macrophages was evident at the 95% confidence level for three of the six time points and was at least at the 85% confidence level for all sampling periods. The glucose microdialysis sampling RR% in the culture wells was also significantly different between the resting and activated macrophages at the 90% confidence level or greater for four of the six time points. The cause of the large variation in the glucose concentrations and, thus, the microdialysis EE% at the 60- and 120-min collection periods is uncertain. 2-Deoxyglucose and Glucose EE in Macrophage Cell Cutlure. Prior to in vivo studies, microdialysis probes were placed into macrophage cultures with added LPS and perfused with 2-DG in a delivery mode. Figure 2 depicts the 2-DG EE% between the resting and LPS-activated macrophages as a function of time. The 2-DG average EE% obtained from the LPS-activated macrophages (63 ( 7%) across all the time points is significantly greater than that from resting macrophages (36 ( 9%) at the 95% confidence level (p < 0.05). The change in 2-DG EE% reflects the alteration in the localized uptake of the infused 2-DG during the microdi-

Figure 2. Localized infusion (microdialysis delivery) of 5 mM 2-DG at 1.0 µL/min through the microdialysis probe to macrophage cultures to which LPS has been added (9) or resting (2) macrophages. The culture media initially contained 5 mM glucose. The term EE% denotes the amount of 2-DG extracted from the infusion fluid into the macrophage culture wells.

alysis sampling process. The reason for the low EE% values for 2-DG at 90 and 150 min are uncertain and may be due to experimental error with this biological system. These in vitro data show that activated macrophages affect microdialysis sampling EE%. This difference in microdialysis sampling EE% is not possible without a significant increase in the macrophage 2-DG uptake kinetics. Although it is not possible to measure the glucose concentration at the probe outer radius, an increase in localized glucose uptake near a sensor surface could affect glucose detection. More importantly, a localized increase in glucose uptake could affect calibration and lead to measurement error, since implanted glucose sensors are calibrated relative to blood values. 2-Deoxyglucose in Vivo Microdialysis. To further test if 2-DG can be used as an internal standard to determine macrophage activation at the implant site, microdialysis probes were implanted into subcutaneous tissue of male Sprague Dawley rats. In each experimental animal, one probe was implanted for a set period of time (3,5, and 7 days) on one side of the dorsal subcutis,

Table 3. Relative Recovery (RR%) and Concentrations of 2-Deoxyglucose and Glucose in Macrophage Cultures timea min 30

60

RR%LPS RR%resting p (t-test)RR% [2-DG]well,LPS,c mM [2-DG]well,resting,c mM

51 ( 5 75 ( 7 0.03†b 5.2 ( 0.2 4.9 ( 0.2

61 ( 6 73 ( 3 0.12 5.1 ( 0.1 4.9 ( 0.2

RR%LPS RR%resting p (t-test)RR% [glucose]well,LPSc [glucose]well,restingc

57 ( 1 72 ( 3 0.02† 5.2 ( 0.3 5.2 ( 0.0

66 ( 9 74 ( 3 0.30 5.2 ( 0.2 5.2 ( 0.0

90

120

150

180

2-Deoxyglucose 54 ( 10 78 ( 9 0.10 5.1 ( 0.2 4.9 ( 0.2

56 ( 8 72 ( 7 0.10 4.9 ( 0.1 4.9 ( 0.1

56 ( 3 75 ( 9 0.04† 5.0 ( 0.1 4.8 ( 0.2

56 ( 5 70 ( 10 0.05† 4.8 ( 0.1 4.8 ( 0.2

Glucose 60 ( 13 79 ( 6 0.09 5.2 ( 0.2 5.1 ( 0.0

64 ( 15 73 ( 11 0.32 5.0 ( 0.2 5.1 ( 0.1

58 ( 5 74 ( 9 0.02† 4.9 ( 0.2 5.0 ( 0.1

62 ( 10 73 ( 11 0.01† 4.6 ( 0.3 5.0 ( 0.1

a Time denotes the time at which a sample was obtained from the microdialysis probes after adding LPS (1 µg/mL) to the culture wells (n ) 3). Microdialysis probes were directly immersed in the well containing both media and macrophages and perfused at 1.0 µL/min. b The symbol, †, denotes a statistically significant difference between the microdialysis RR% from resting as compared to LPS-activated macrophages at the 95% confidence level. c Measurements of 2-DG and glucose concentrations from the wells were performed by removing media that bathes the macrophages that are attached to the culture plate. Data represent mean ( SD.

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Figure 3. 2-DG EE% (left) and glucose concentrations (mM) (right) collected from dialysates from acute (9) and 3-day (2) implanted PC probes placed into the subcutaneous space of anesthetized rats (n ) 3). The probes were infused with 5 mM 2-DG at 1.0 µL/min. Symbols and error bars denote means ( SD

Figure 4. 2-DG EE% (left) and glucose concentrations (mM) (right) collected from dialysates from acute (day 0) (9, 0) and 5-day (2, 4) PC probes implanted into the subcutaneous space of anesthetized rats (n ) 2). The probes were infused with 5 mM 2-DG at 1.0 µL/min. Individual symbols denote values from each animal.

and another probe was acutely implanted as a control on the opposite side on the day of sample collection. The 3-day implantation experiment was performed using three animals; the 5-day and 7-day implantation experiments were each performed using two animals. Figure 3 illustrates the average 2-DG EE% and glucose concentrations obtained from probes implanted for 3 days vs those implanted the day of sample collection paired within the same animal (n ) 3 animals). The in vivo 2-DG EE% values and glucose concentrations at 5 and 7 days compared to the control values are presented in Figures 4 and 5, respectively. Table 4 shows the average 2-DG EE% values and glucose concentrations across all the time points for the acute vs chronic implanted dialysis probes at 3, 5, and 7 days. The assumption in these experiments is that a steady-state EE% was achieved during the microdialysis sampling process. This assumption was confirmed by comparing dialysates obtained after the first 15 min of implantation using a 1.0 µL/min flow rate with those obtained at the first 30-min collection period (after this initial flush period). Among these dialysates, there were no differences in the 2-DG EE% or the glucose concentration (data not shown). Therefore, the six collected dialysates over the 3-h sampling period for each probe were averaged and compared. Table 4 shows that except for a few outliers, the 2-DG EE% and glucose concentrations

between the acute and chronic implanted probes were not statistically different at the 95% confidence level. Summary of in Vitro and in Vivo Data. Macrophages begin to accumulate at implanted surfaces within a few hours and can remain at the site throughout the implant lifetime.14 On the basis of this information, glucose uptake could be altered during the early period of the inflammatory response at an implant site. The in vivo 2-DG EE% values vary significantly compared to those in vitro. For the in vivo studies, there are few differences in 2-DG EE% and glucose consumption between probes implanted acutely vs those implanted for either 3, 5, or 7 days. These observed differences between in vitro and in vivo 2-DG EE% are likely due to the degree of the inflammatory response as well as a potential difference in macrophage cell density per unit volume. The addition of lipopolysaccharide greatly activates cells in culture to an inflammatory state, potentially making the in vitro cells far more metabolically active than those in vivo. The sensitivity of the dialysis probe toward small external metabolic changes could come into play in these studies. We have previously shown that for an in vitro enzyme system, a 20-fold difference in enzymatic activity was required to obtain a statistically significant alteration in observed EE% differences for a substrate.44 This is an important point for the in vitro experiments, since Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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Figure 5. 2-DG EE% (left) and glucose concentrations (mM) (right) collected from dialysates from acute (day 0) (9, 0) and 7-day (2, 4) PC probes implanted into the subcutaneous space of anesthetized rats (n ) 2). The probes were infused with 5 mM 2-DG at 1.0 µL/min. Individual symbols denote values from each animal.

Table 4. In Vivo 2-DG Delivery (EE%) and Glucose Concentrationsa Rats EE%b of 2-DG

glucose concn [mM]

Acute 38 ( 6 32 ( 8 32 ( 7

3-Day Implantation Day 3 Acute 45 ( 3 5.5 ( 0.4 41 ( 5 † 5.4 ( 1.1 39 ( 10 4.7 ( 0.7

Day 3 6.6 ( 0.6†c 5.6 ( 0.9 4.2 ( 0.5

Acute 45 ( 4 29 ( 4

5-Day Implantation Day 5 Acute 44 ( 4 3.9 ( 1.0 40 ( 4† 5.8 ( 1.0

Day 7 3.2 ( 0.3 4.8 ( 1.4†

Acute 41 ( 6 31 ( 10

7-Day Implantation Day 7 Acute 36 ( 4† 5.3 ( 1.3 34 ( 8 6.4 ( 1.0

Day 7 4.6 ( 0.3 5.6 ( 0.6

a Probes were implanted into the subcutaneous space and infused with 5 mM 2-DG at 1.0 µL/min over a 3-h period with samples collected every 30 min while the animal was maintained under anesthesia. b EE% denotes percent 2-DG extracted from the probe into the tissue and was averaged over the six samples obtained during the 3-h period. Data represent mean ( SD, n ) 6. c The symbol, †, denotes a statistically significant difference between the acute and chronic implant 2-DG microdialysis EE% at the 95% confidence level.

statistically significant differences in RR% and EE% were observed, suggesting a very large change in metabolic activity, despite the moderate change in the extraction efficiency values. It should be noted that differences in product concentrations collected into microdialysis sampling probes as a function of time are observable for small changes in enzymatic activity external to a probe. However, in these studies, 2-DG is trapped inside the cell. Whereas the phosphorylated metabolite (2-DG-6-phosphate) that accumulates in cells would be a reasonable target in vitro for measuring metabolism differences, it is not possible to measure this product in vivo, since it is not released into the extracellular fluid space.45 (44) Steuerwald, A. J.; Villeneuve, J. D.; Sun, L.; Stenken, J. A. J.Pharm. Biomed. Anal. 2006, 40, 1041-1047. (45) Sasson, S.; Oron, R.; Cerasi E Anal. Biochem. 1993, 215, 309-311. (46) Wisniewski, N.; Klitzman, B.; Miller, B.; Reichert, M. J. Biomed. Mater. Res. 2001, 57, 513-21.

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In addition to activated macrophages, other tissue processes can affect EE% during a long-term microdialysis probe implant, including biofouling of the membrane and tissue mass transport resistance changes caused by the foreign body response.46 It is known that for small molecules, the tissue resistance contributes most to the variation in the analyte EE%. It is interesting to note that among the 3-, 5-, and 7-day implants, there was little difference in the 2-DG EE%, as compared to the probes implanted as controls. In some cases, the longerterm implanted probes exhibited higher delivery than the acute implant. This could potentially be caused by an edema that would create additional wound fluid around the device and would serve to decrease the analyte mass transfer resistance through the tissue (e.g., the volume fraction increases). CONCLUSIONS The IC-PAD chromatographic method coupled with the macrophage cell line provided a straightforward method to measure glucose uptake alterations near the microdialysis probe. The effects of the foreign body response on analyte collection are an integrated whole that have not previously been systematically studied. This work shows that macrophages in vivo appear to be less reactive toward the microdialysis probe than those activated in vitro. Additionally, no significant differences in either 2-DG EE% or glucose concentrations were observed among microdialysis probes implanted for 3, 5, and 7 days. ACKNOWLEDGMENT We thank Dr. Michelle Lennartz and Dr. Daniel Loegering at the Center for Cell Biology and Cancer Research and the Center for Cardiovascular Sciences at the Albany Medical College for their guidance with cell culture methodology and help with some of the animal experiments. We gratefully acknowledge the support from NIH EB001441. The helpful comments from all the manuscript reviewers are also gratefully acknowledged.

Received for review June 21, 2006. Accepted September 15, 2006. AC061124I